Metabolism of Anabolic Steroids in Man: Synthesis and Use of Reference Substances for Identification of Anabolic Steroid Metabolites
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Anaiytzca Chumca Acta, 275 (1993) 23-48 23 Elsevler hence Publishers B V , Amsterdam Metabolism of anabolic steroids in man: synthesis and use of reference substances for identification of anabolic steroid metabolites Will1 Schanxer and Manfred Domke LkutscheS’rthochschuk Koln, Inrhtut f?u Bwchemre, Carl-L&m-Weg 4 5ooO Cologne 41 (Germuny) (Recewed 20th May 1992) AhStl-& The use of anabohc steroids was banned by the International Olympic Comnuttee for the first tune at the Olympic Games m Montreal m 1976 Since that tie the nususe of anabohc steroxls by athletes has been controlled by analysis of urme extracts by gas chromatography-mass spectrometry @C-MS) The excreted steroids or their metabohtes, or both, are isolated from urme by XAD-2 adsorption, enzymatx hydrolyss of coqlugated excreted metabobtes anth /?-glucuromdase from Es&en&u co& bqmd-bqmd extractlon mth diethyl ether, and courted mto tnmethylsdyl (‘INiS) derwatwes The confirmation of an anabobc stenxd nususe ts based on comparison of the electron nnpact lomzation (EI) mass spectrum and GC retention tie of the isolated steroid and/or its metabohte with the El mass spectrum and GC retention time of authentic reference substances For this purpose excretion studies with the most common anabobc steroids were performed and the mam excreted metabobtes were synthesued for bolasterone, boldenone, 4-chiorodehydromethyltestosterone, clostebol, drostanolone, tluoxymesterone, forme- bolone, me&a&one, mesterolone, metandlenone, methandnol, metenolone, methyltestosterone, nandrolone, norethandrolone, oxandrolone and stanozolol The metabobsm of anabohc Hero& the synthesis of theu mam metabohtes, theu GC retentmn and EI mass spectra as TMS dewames are dIscussed &ywor& Gas chromatography, Gas chromatography-mass spectrometv, Anabobc steroids, Metabohsm The misuse of testosterone and anabohc abohc steroids and then metabohtes are isolated steroids by athletes to improve their performance from urme, derrvatrzed wrth N-methyl-N-tnmeth- has led to a ban on anabohc steroids by the ylstlyltnfluoroacetarmde (MSTPA) and analysed International Olympic Comnnttee (IOC) and na- by gas chromatography (GC) and mass spectrom- tional and mtematronal sports federations An- etry (MS) usmg electron impact (ED romxatron at abohc steroids were banned for the first time at 70 eV Because most anabohc steroids are com- the Olympic Games 1976 m Montreal and testos- pletely metabohxed and no parent steroid is ex- terone at the Olympic Games 1984 m Los Ange- creted, the metabolism of the anabohc steroids les To control the misuse of an anabohc steroid must be known Papers on the metabolism of the urmes of athletes are collected directly after anabohc sterords m man were available for bolde- competrtron or out of competrtron and have to be none [1],4-chlorodehydromethyltestosterone [2,3], analysed by IOC accredited laboratories The an- metandrenone [4,5], methyltestosterone [6], nan- drolone [71 and norethandrolone [8] The identifi- catron of these metabohtes was based on the Comzvpondence to W Schanzer, Deutsche Sporthochschule Koln, Instltut fir Bmchenue, Carl-Diem-Weg 6,5ooO Cologne isolation and purrficatron of urmary excreted 41 (Germany) metabohtes after application of large amounts of 0003~2670/93/$06 00 Q 1993 - Elsevler Science Publishers B V All nghts reserved 24 W Schanzer and M Lkwukz/Am! Chm Acta 275 (1993) 23-48 the anabohc steroid and characterlzatlon by melt- mmmm hydnde, chromnun tnoxlde, platmum mg points and mfrared (IR), ‘H nuclear magnetic dloxlde and 10% palladium on charcoal were resonance (NMR) and mass spectrometry At purchased from Aldrich (Stemhelm) Bolasterone least the proposed structure of the metabohte was purchased from UpJohn, Cchlorodehydro- was elucidated by its synthesis methyltestosterone was a gift from Jenapharm GC with fused-s&a capdlary columns corn- (Jena), fluoxymesterone was a gift from Cuba- bmed with MS with EI lomzatlon allows very fast Geigy, furazabol was a g& from Danchl (Tokyo), and relatively simple sample preparation to iso- oxandrolone was a gti from Searle, oxymesterone late small amounts of anabolrc steroids and their was a @ft from Carlo Erba, mesterolone, metabohtes from urine [9-113 The isolated metenolone and metenolone acetate were g&s metabohtes are characterized by then GC reten- from Schermg (Berlin), formebolone was a gift tion times and EI mass spectra A metabohte is from A H Beckett (London), drostanolone propl- identified when the companslon wth an authen- onate was a gift from Grunenthal (Aachen), tic reference substance shows the same GC reten- boldenone, clostebol acetate, mestanolone, tion time and EI mass spectrum metandlenone, methandnol, methandrlol dlpro- As most of the anabohc steroid metabohtes plonate, methyltestosterone, nandrolone and were not commercially avalable m earlier times, stanozolol were purchased from Sigma (Delsen- so-called “reference substances” were obtained dorf), norethandrolone, 6&hydroxytestosterone from urme m excretion studies vvlth anabohc and 6/3-hydroxyandrostenedlone were purchased steroids A cntlclsm of this method was the low from Steralolds via Paesel (Frankfurt) accuracy of a urinary “reference substance” for N-methyl-N-trlmethylsllyltrlfluoroacetamlde which the structure 1s not exactly known and the (MSTFA) was synthesued m the laboratory All possible nnpur?ty of the urmary extract other reagents and solvents were of analytlcal-re- To circumvent this disadvantage we started m agent grade 1987 the synthesis of the mam metabohtes of those anabohc steroids which were mainly mls- Metabohsm studws used m sports The synthesis of metabohtes of Metabohsm studies were performed by oral boldenone [ 121,metandlenone [ 131, stanozolol [ 141 application of the anabohc steroids I-XX to male and the synthesis of 17-epunenc steroids [151 volunteers and by self-admmlstratlon Usually a have been published Further pubhcatlons are m smgle dose of 20 mg of steroid was applied and preparation the followmg urine samples were collected and This paper gives an oveMew of the mam stored at 4°C metabolic pathways, the synthesis of the mam metabolrtes of anabohc steroids and their tnmethylsllyl (TMS) derlvatlzatlon for GC-MS Isolatwn of an&&c steroids and theu metabo- and presents the GC retention mdlces and EI htes mass spectra of the anabohc steroids excreted The steroids and/or then metabohtes were unchanged and their mam metabohtes, which are isolated as described by Domke et al [9] wth routmely used for screemng and confirmation some modifications The actual sample prepara- The pharmacokmetics of the excreted steroids tion 1s described and metabohtes will not be discussed Isolatwn of u?aconJugatedexcreted stero& To 5 ml of urme are added 0 5 g of sodium EXPERIMENTAL hydrogencarbonate-potassium carbonate (2 1, w/w> solid buffer and 50 ng of 4a-hydroxy- Steroids and chermcals stanozolol as mtemal standard and the uncon~u- K-Selektrlde (1 M potassmm tn-set -butyl- gated steroids are extracted with 5 ml of dlethyl borohydnde m tetrahydrofuran), hthmm alu- ether (dlstllled over calcium hydride) The ether W SchamerandM Donke/AnalChm Acta275(1993)23-48 25 layer IS transferred after centrrfugatlon and evap- Column C was used (see GC-FID parameters) orated to dryness under vacuum The column temperature was programmed from See also the lsolatlon of conlugated steroids 2ooOC at 4O”C/mm to 320°C (held for 3 mm) The mjector temperature was 300°C and the interface Isolatwn of conlugated excreted sterouii and temperature 3u)“C metabohtes ConJugated fractwn The instruments and car- To 2 ml of urme 1s added 1 pg of methyl- rier gas were the same as above Column A was testosterone (mternal standard) and the steroids used (see GC-FID parameters) The column are adsorbed on Amberhte XAD-2 polystyrene temperature was programmed from 180°C at resin The XAD-2 columns (Pasteur pipette, CC/mm to 240°C and at 15”C/mm to 320°C closed with a glass pearl, bed height 2 cm) are The injector temperature was 300°C and the m- washed with 2 ml of doubly dlstllled water and terface temperature 320°C eluted wth 2 ml of methanol The methanohc eluate 1s evaporated to dryness and the residue 1s GC-FID parameters dissolved m 1 ml of 0 2 M sodium phosphate An HP 5880 gas chromatograph was used The buffer (pH 7) carrier gas was hehum at 15 ml/mm at 18o”C, At this stage a separation of conjugated and splitting ratio 1: 10, pressure 27 PSI The column unconjugated excreted steroids 1s possible The temperature was programmed from 180°C at unc.onJugated steroids can be extracted with 5 ml 5”C/mm to 320°C Column A was Hewlett- of dlethyl ether The remammg ether must be Packard Ultra-l fused s&a, cross-linked methyl- removed by evaporation of the buffer solution for silicone (OV-11, 17 m X 0 2 mm 1 d , film thrck- a short time To the buffer solution 50 ~1 of ness 0 11 pm Columu B was Hewlett-Packard P-glucoromdase from E cob (K12, Boehrmger, HP-1 fused slhca, cross-linked methyls&cone Mannheun) are added and after 1 h at 50°C the (OV-0, 17 m x 0 25 mm I d , film thickness 0 33 buffer solution 1s made alkalme (pH 9-10) with pm Column C was Chrompack WCOT fused 250 ~1 of 5% potassmm carbonate solution and slhca CP-SIL 8CB, cross-lmked 5% phenyl- the steroids are extracted mth 5 ml of dlethyl methyls&one (SE-54), 17 m x 0 25 mm 1 d , film ether on a vlbro mixer for 30 s After centrtiga- thickness 0 25 pm Column D was Macherey- tlon the ether layer 1s transferred and evaporated Nagel Permabond, fused silica, cross-lmked 5% to dryness under vacuum phenyl-methylsdlcone (SE-541,